Waterborne pathogenic microorganisms are a major source of disease worldwide. Despite measures instituted to ensure the microbiological safety of drinking water, disease-causing microorganisms are regularly transmitted via water supplies. The principal waterborne bacteria of concern are Salmonella spp., Shigella spp., Yersinia spp., Mycobacterium spp., enterocolitica, as well as Escherichia coli, Campylobacter jejuni, Legionella, Vibrio cholerae. These bacteria range from 0.5 to several microns and are either ovoid (cocci) or rod-like (bacillus) in shape. Cryptosporidium and other protozoa are about 3-5 microns in size and are resistant to many forms of chemical disinfection. Cryptosporidium has been responsible for several major pollution events and many deaths.
The EPA Science Advisory Board ranks viruses in drinking water as one of the highest health risks. Waterborne pathogenic viruses include Enteroviruses, (such as polioviruses, Coxsackievirus (groups A and B), echoviruses, hepatitis A virus), rotaviruses and other reoviruses (Reoviridae), adenoviruses, and Norwalk-type agents. The numbers of viruses detected per liter of sewage range from less than 100 infective units to more than 100,000 infective units. In some instances, the ingestion of a single infectious unit can lead to infection in a certain proportion of susceptible humans. Constant exposure of large population groups to even relatively small numbers of enteric viruses in large volumes of water can lead to an endemic state of virus dissemination in the community. Currently, there are no EPA regulations mandating virus removal, due to the fact that the hazard has not been adequately quantified. There are, however, military specifications requiring the removal of viruses (as Hepatitis)>4 logs (>99.99%) as well as bacteria (as E. coli)>5 log (99.999%) and Cryptosporidium>3 logs (99.9%).
The principal disinfection methods are chemical oxidation and micro filtration. Chemical oxidants include ozone, chlorine and chlorine derivatives. Viruses are more resistant to environmental conditions and sewage treatment processes, including chlorination and UV radiation, than many of the sewage-associated bacteria. In laboratory studies, enteric viruses survive for up to 130 days in seawater, surpassing those reported for coliform bacteria.
There is growing concern about the toxicity and potential carcinogenicity of disinfectant by-products (DBP's) that form as a result of chemical treatment. In public water supplies, it has not been cost effective to substitute filter sterilization because the flow rate of filters rated for virus removal is far too low to be practical. Further, because pathogenic bacteria can proliferate in piping, filtration at the point of use is preferable. Point of entry (POE) and point of use (POU) purification systems based on reverse osmosis (RO) filtration or ultraviolet treatment (UV) are generally used for removing microbial pathogens from municipal water as well as from untreated ground water sources. Both RO and UV systems are limited by low flow rates and require water reservoirs to sustain effective flow when taps are open. In addition, complete sanitization by UV is uncertain because pathogens may be shielded by colloidal particles. Thus, filtration is a necessary adjunct. Both RO and UV systems tend to be rather expensive for a small POU (single faucet) system and are very complex, requiring extensive service for maintenance. A filter capable of removing all microbial pathogens is of great value. Further, it is a major economic advantage to produce a filter that can be installed with as much as ease as installing current chemical filters.
Purifying water is also important in medical and dental offices. Dental-unit water systems (DUWS) harbor bacterial biofilms that serve as a haven for pathogens that often exceed dental association standards. The pathogens found include many serious species such as Legionella pneumophila and contamination of water lines with viruses and potentially HIV or Hepatitis virus could occur from back siphoning of fluids from prior patients. A POU filter capable of sanitizing DUWS water by removing bacteria and virus at high flow rate is also desired for dental suites.
Colloidal contamination of water can contribute to turbidity (cloudy appearance) of water. Such colloidal particles generally include organic matter such as humic material, pathogens, as well as nano size inorganic minerals. Colloids provide sorption sites for microbes, pesticides, other synthetic organic chemicals, and heavy metals. Their nano size dimensions tend to clog filter systems.
Improved technology is needed in the detection and identification of pathogens and particularly viruses because methods of concentrating them are not efficient. Low cost samplers of virus are needed for assaying surface waters. While such filters need not be sterilization grade, the collector should be capable of removing the bulk of virus from a 500-1000 liter water sample within two hours. Then the virus on the filter must be eluted intact and viable for subsequent analysis. AMF Cuno's MDS-1 filter is predominant in the analysis of environmental water for virus. Unfortunately such filters are cost prohibitive for routine use, particularly since these are single use filters. The price for single use filters must be significantly reduced before the EPA would consider virus sampling as a routine procedure. Recently, potential terrorist use of biological weapons (BW) in air and water has increased the need for early detection and measurement. Effective detection is difficult due to the limited sensitivity of current bio-analytical methods, particularly with virus that are much smaller, more difficult to concentrate and more likely to be pathogenic. Tens to hundreds of liters of air or water need to be sampled to provide sufficient particles for detection in a sample. Particle collectors for air sampling are limited to cyclone separation and impactors and are impractical for virus size particles.
Fibrous depth filters retain particles principally by impaction of the particle onto the fiber where they adsorptively adhere while membranes retain particles principally by size exclusion. Membranes are available with pore sizes small enough to sieve out bacteria and viruses, but fibrous depth filters are incapable of sanitizing water. In the context of filtration separations, pore sizes in reverse osmosis membranes extend from about 1 to 10 Å to 20 Å, ultra filtration from about 1 nanometer (10 Å) to 200 nm, micro filtration from about 50 nm or 0.05 micron to about 2 microns, and particle filtration from about 1 to 2 microns and up. As the size of the pathogen decreases, the pore size of the filtration membrane must be reduced. This results in a drastic pressure drop and reduces process flow rates. Many viruses are as small as 30 nm and commercially available membranes are limited to approximately 2 to 3 log reduction for such small particles [Willcommen]. Membranes are susceptible to clogging, and pre-filters must be used to remove coarser particles to extend the life of the more expensive membrane. Furthermore, membranes are susceptible to point defects such as overlapping pores and finger voids that greatly affect reliability.
Virus must be removed from proprietary medicinal products as well as human blood and plasma-derived products. FDA has recommended that all purification schemes use one or more ‘robust’ virus removal or inactivation steps. Robust steps were defined as those that work under a variety of conditions and include low pH, heat, solvent-detergent inactivation and filtration. Virus removal via heat, chemical, ultraviolet or gamma radiation could denature sensitive proteins and further require the removal of said chemical agents or denatured proteins. Accordingly, filtration is regarded as a preferred method of virus removal.
In recent years there has been a remarkable expansion of biotechnology including the synthesis of proteins formed via recombinant methods, removal of proteins from blood plasma, modified hemoglobin products, mammalian serum products as well as protecting fermenters from contamination by viruses. Prions are proteins suspected of causing Creutzfeldt-Jacob disease (CJC). They are about 10 mm in size, thus, smaller than virus. In pharmaceutical manufacture, micro porous and ultra porous membranes are used to purify incoming process water streams as well as control effluents and by-product streams to assure that there is no contamination of water discharges.
Originally 0.45 micron filters were regarded as “sterilization rated” based on their ability to efficiently retain Serratia marcescens organisms, but studies showed that the filter could be compromised. A smaller bacteria Pseudomonus diminuta (P. diminuta) (0.3μ) is now widely used for testing filter integrity, and a 0.22μ pore filter is now accepted as sterilization grade in removing bacteria. Ultra porous membranes, with pore sizes as small as 20 nm are used for filtering virus. Nano porous membranes such as used in reverse osmosis are capable of filtering virus, but with even further increases in flow restriction. In the manufacture of proteins, regulatory authorities suggest that sterilization from viruses should require a virus reduction factor (LRV) of at least 12 orders of magnitude. This value is calculated to result in less than one infectious particle per 106 doses. Such a level of virus removal currently requires multiple step processes.
Packed beds containing multivalent cations such as Al3+ and Mg2+ at a pH of about 3.5 may retain viruses. Lukasik et al. were able to remove viruses efficiently from raw sewage laden water over significant time using deep beds of sand modified with ferric and aluminum hydroxides. Farrah incorporated metallic oxides of aluminum and other metals into diatomaceous earth and achieved significant improvement in adsorption of viruses. Farrah and Preston improved the adsorption of several viruses by modification of cellulose fiber filters with flocculated ferric and aluminum hydroxides.
Filters may be constructed from a wide variety of materials. While glass fiber filters tend to be weak, a number of chemical binders will improve their physical properties and modify their chemical characteristics. Ceramic filters often have desirable combinations of properties, but they can be brittle. Metal filters often overcome this limitation, but they can be expensive. The benefit to using ceramic and metal filters is that they are often cleanable and reusable. Filter fibers or membranes are produced from a variety of polymers including: polyvinylidene difluoride (PVDF), polyolefin and acrylics. Sorbents such as granular silica, diatomaceous earth or granular carbon have also been incorporated into filter media.
Electro kinetic forces aid the capture of particles from water. Hou has described a qualitative picture of filtration by electro kinetic phenomena, where the phenomenon is explained by concepts similar to those in colloidal chemistry. If the electrostatic charges of the filter media and particulates are opposite, electrostatic attraction will facilitate the deposition and retention of the particles on the surface of the filter media. If they are of similar charge, repulsion will occur and deposition and retention will be hindered. The surface charge of the filter is altered by changes in pH and the electrolyte concentration of the solution being filtered. This phenomenon is explained by the electric double-layer theory of colloidal chemistry. A particle immersed in an aqueous solution develops a surface charge by adsorbing ions on its surface. A fixed layer of oppositely charged ions develops around the surface of the filter. To maintain the electrically neutral system, there is a diffused layer containing a sufficient number of counter-ions extended for some distance into the solution. If the bulk solution of counter-ions increases by addition of cationic salts or increasing pH, the thickness of this layer decreases because less volume is required to contain enough counter-ions to neutralize the surface charge. The reduction of thickness of this layer facilitates the approach of the two surfaces, allowing Van der Waals forces to take effect. Lowering the pH or adding cationic salts thus reduces electronegativity, and allows for some adsorption to occur under these conditions. Virus adsorbance is facilitated in most natural and tap water, having pH ranging between about 5-9 [Sobsey]. Acid or salt addition is often needed to effect virus removal by electronegative filters. Hou claims that electropositive filters have widespread application in the removal of microorganisms from water, for the concentration of both bacteria and viruses from water and harvesting for removal of endotoxins from contaminated partenterals and foods, and for immobilization of microbial cells and antigens.
Fibrillated asbestos, having an electropositive surface charge and a surface area of approximately 50 m2/g was used extensively as cellulose/asbestos filter sheets for filtering pathogens. The fibers of asbestos produced a very fine pore structure that was capable of mechanical straining as well. However, concerns about the health hazards of asbestos terminated its use, and efforts began to develop an asbestos substitute. These efforts included attempts to chemically modify the surfaces of hydrophobic polymeric filter materials to produce coatings with electropositive charges.
For example, U.S. Pat. Nos. 4,007,113; 4,007,114; 4,321,288 and 4,617,128 to Ostreicher, describe the use of a melamine formaldehyde cationic colloid to charge modify fibrous and particulate filter elements. U.S. Pat. Nos. 4,305,782 and 4,366,068 to Ostreicher, et al. describe the use of an inorganic cationic colloidal silica to charge modify such elements. In U.S. Pat. No. 4,366,068, the “fine” silica particle exhibits an average particulate dimension of less than about 10 microns and is coated with at least 15% alumina. U.S. Pat. No. 4,230,573 to Kilty, et al. describes the use of polyamine epichlorohydrin to charge modify fibrous filter elements, see also U.S. Pat. No. 4,288,462 to Hou, et al., and U.S. Pat. No. 4,282,261 to Greene. Preferred methods of making filter media are described in U.S. Pat. No. 4,309,247 to Hou, et al. and are being sold by Cuno, Inc. under the trademark ZETA PLUS. Similar attempts at cationic charging of filters were made in U.S. Pat. Nos. 3,242,073 and 3,352,424 to Guebert, et al., and U.S. Pat. No. 4,178,438 to Hasse, et al. U.S. Pat. No. 5,855,788 to Everhart describes a method of modifying the surface of filters based on woven or non-woven fabrics, or aperture polymers by adsorbing amphiphilic protein such as derived from milk. The protein is modified with metal hydroxides such as alumina derived from sol-gel reactions. The filters remove waterborne pathogens primarily by chemical and electrokinetic interactions rather than by sieving. A log 3 reduction was obtained for Vibrio cholerae bacteria. The inventors observed that filters having modified surface charge characteristics have different filtration efficiencies for different types of waterborne pathogens, such as, for example, different types of bacteria.
U.S. Pat. No. 5,085,784 to Ostreicher proposes a charge modified filter media comprising cellulose fiber, silica based particulate, and a cationic water-soluble organic polymer. The polymer is adsorbed onto the filter that includes an epoxide and a quaternary ammonium group, capable of bonding to a secondary charge-modifying agent. The modifying agent is preferably an aliphatic polyamine. U.S. Pat. No. 4,523,995 to Pall describes a filter media prepared by mixing glass, with polyamine-epichlorohydrin resin, to form a dispersion. A precipitating agent is added to the dispersion to precipitate the resin and coat the microfibers. The preferred precipitating agents are high molecular weight polymers containing anionic charges. The resulting coated microfibers are described as having a positive zeta potential in alkaline media and enhanced particulate removal efficiencies for fine particulate removal, including bacteria and endotoxins (pyrogens). However, Robinson et al., Mandaro and Meltzer describe the limitations of prior art cationic charge modified media in terms of general loss of filtration performance at high pH and, the inability of such media to achieve effective removal of very fine particle and/or pyrogens (endotoxins) removal.
Cationically charged membranes, used for the filtration of anionic particulate contaminants, are also known and described in U.S. Pat. No. 2,783,894 to Lovell and U.S. Pat. No. 3,408,315 to Paine. U.S. Pat. Nos. 4,473,475 and 4,743,418 to Barnes, et al. describes a cationic charge-modified micro porous nylon membrane having a charge-modifying amount of an aliphatic amine or polyamine, preferably tetraethylene pentamine bonded to the nylon. U.S. Pat. No. 4,604,208 to Chu, et al. describes an anionic charge modified nylon micro porous filter membrane. The charge-modifying system is a water-soluble polymer capable of bonding to the membrane and anionic functional groups such as carboxyl, phosphorous, phosphonic, and sulfonic groups. U.S. Pat. Nos. 4,473,474; 4,673,504; 4,708,803 and 4,711,793 to Ostreicher, et al., describe a nylon membrane charge modified with epichlorohydrin-modified polyamide having tertiary amine or quaternary ammonium groups, and a secondary charge-modifying agent that may be an aliphatic polyamine. Cationic charge modified nylon membranes covered by these patents to Ostreicher, et al. and Barnes, et al. are now being sold by Cuno, Inc., under the trademark ZETAPOR. Positively charged modified microporous filter media are available from Pall Corp., that uses nylon 66 or a positively charged polyethersulfone sulfate membrane. Micropore Corp. produces a charge-modified poly vinylidene difluoride (PVDF) membrane.
Filters are used generally in two different modes. In the depth or dead end filter, all the fluid flows through the membrane or media. In the cross-flow (tangential) flow filter the feed flow is axially channeled, while pure liquid (permeate) flow through the filter media. This type of filter limits the thickness of the filter cake making and allowing greater flowrate, while in conventional dead-end filtration, the filter cake increases with time, resulting in pressure drops that cause cessation of flow. Filtration speed is important in virtually all industrial processes including pharmaceutical manufacturing, biological processing, and laboratory experimentation. Membranes used in filtering sub-micron to nanometer size particles have very low flux unless compensated for by substantial increases in pressure on the filter or by increases in filter membrane area. Increasing pressure or filter area markedly increases capital and operating costs. Clogging further degrades the low flux of small pore membranes.
Pyrogens are substances that contain endotoxins that are fever-inducing substances. Endotoxins are high molecular weight (between 10,000 to 1 million) complexes, which derive from gram-negative bacteria that shed their outer membrane into the environment, causing fever in humans. The endotoxin is not affected by autoclaving because it is stable for several hours at 250° C. Endotoxins can, however, be removed by reverse osmosis but RO processing is difficult because of the small membrane pore size. Moreover, desirable substances such as salts are excluded by reverse osmosis and this is a drawback in forming non-pyrogenic parenteral solutions. U.S. Pat. No. 5,104,546 describes a ceramic ultra-filter that is capable of separating pyrogens from parenteral fluids. The ceramic ultra-filter is a zirconium oxide layer over an alumina ceramic having a nominal pore size of about 5 namometers. The filter is capable of separating pyrogens to the extent of 5 logs, however significant driving pressure (80 psi) and a cross-filter length of 20 cm are required to produce an initial flux of only 50 L/hr/m2/atmosphere.
New analytical schemes are being developed for pharmacological or biochemical materials, where specific reagents are retained onto high surface area substrates. Membranes are also commonly used as supports for diagnostic assays like electrophoresis, cytology of fluids, and DNA hybridization. Many analytical methods involve immobilization of a biological binding partner of a biological molecule on a surface. The surface is exposed to a medium suspected of containing the molecule, and the existence or extent of molecule coupling to the surface-immobilized binding partner is determined. For instance, in U.S. Pat. No. 5,798,220 a native macromolecule is bound to a support surface and used in an immunoassay to screen biological fluids for antibodies to the macromolecule in its bound state. U.S. Pat. No. 5,219,577 describes a synthetic biologically active composition having a nanocrystalline core. U.S. Pat. No. 6,197,515 describes for a method of capturing a biological molecule, for example at a biosensor surface, by exploiting biological binding interactions. A substrate that would efficiently bond the first biological molecule would facilitate such analytical strategies. Nucleic acids are sorbed to metal oxide supports including alumina to enable the resulting compositions to be used for hybridizing, labeling, sequencing, and synthesis.
Electrophoresis is one of the most widely used separation techniques in the biologically related sciences. Molecular species such as peptides, proteins, and oligonucleotides are separated as a result of migration in a buffer solution under the influence of an electric field. This buffer solution normally is used in conjunction with a low to moderate concentration of an appropriate gelling agent such as agarose or polyacrylamide to minimize the occurrence of convective mixing. The highest resolution is obtained when an element of discontinuity is introduced in the liquid phase. Elements such as pH gradients, or the sieving effect of high-density gels have a great influence on the separation of different size molecules. Membrane barriers may also be introduced into the path of migrating particles. High surface area electropositive absorbers, if added to such gels, can enhance separation factors.
The “Genetic Revolution” is creating a need for technologies related to bio-separation. Because recombinant retrovirus, sadenovirus of adeno-associated virus vectors offer some of the best vehicles for accomplishing efficient transfer and integration of genetic material, there is a clear need for virus isolation methods which are both effective and cause little or no damage to the viral particles. Density gradient centrifugation has always played an important part in concentration and purification of virus particles but the gradient media used most prominently, for example, sucrose and CsCl, pose a number of problems. Both media are highly hyperosmotic and the entities used to bond viruses generally have to be removed by ultra filtration prior to further processing or analysis. Moreover, a precipitating agent that would remove virus by precipitation, or efficient low-pressure drop filtration, would facilitate such processing. [Nycomed]
Separation of macromolecules such as proteins is a considerable cost in the manufacture of pharmacological products. Chromatography has been used for decades to perform biological separations. In chromatography, a mixture is applied in a narrow initial zone to a stationary sorbent and the components are caused to undergo differential migration by the flow of the liquid. In the case of ion chromatography, that differential migration is caused by the divergent attraction of charged molecules in an oppositely charged stationary phase. Chemically modified cellulose containing silica is used for the stationary phase in the manufacture of commercially important biomolecules in the food, biopharmaceutical, biotechnology and pharmaceutical industries. For example, such media used in many different large-scale processes for the manufacture of antibodies, enzymes, peptides, plasmids and hormones. Alternative stationary phases can include metals and metal oxides, for example, particulate aluminum oxide. Membrane absorbers (MA) are membranes with functionalized surface sites for chromatography. The resolution of traditional chromatography beads is inversely proportional to the size of the beads and MA devices produce very high resolution because they have higher external surface area compared to granules. A fibrous media capable of such resolution having lower flow restrictions would provide even more rapid and economical separation, for both purifying solutes and for analytical purposes.
Fine aluminum hydroxide gels and sols have been used as precipitation aids and as powdered sorbents for organic macromolecules. For example, Al(OH)3 is used bind vitamin K factors. Nucleic acids are often contaminants in solutions of protein and can be precipitated by exposure to nucleases, [Ahuja, p. 368] shear, low ionic strength or high pH. Most of these methods affect resident proteins and have a relatively low degree of selectivity. Because nucleic acids possess negatively charged phosphate residues, precipitating agents with positively charged groups have the ability to selectively remove of nucleic acids from solution. This principle has given rise to the search for several precipitating agents for nucleic acids.
Cells are often cultured in reactors to produce biological and pharmacological products. Such cells can be bacterial or mammalian. In order to maintain a cell culture, oxygen and other nutrients generally must be supplied to the cells. Cell cultures are usually maintained in reactors by perfusion, wherein a cell culture medium, including oxygen and other nutrients, is directed through the cell-culture reactor. Cell-culture reactors, however, can support only small cell loadings per unit of reactor volume. They can only operate within a small window of flow or agitation rates. Porous substrates that would immobilize cells while allowing greater nutrient and dissolved gas exchange, would allow greater reactor loading. Similarly, biocatalytic reactions are performed in reactors, where an enzyme catalyst is retained to a porous inorganic support. Immobilization enhances the enzymes thermal and chemical robustness, while maintaining a high catalytic activity over a wide range of environmental conditions. A high surface area electropositive support would retain and immobilizes enzymes, providing for more rapid chemical reaction.
Ultra-pore water is used in a number of industries. Membrane ultra-filters are used to produce high purity water for the fabrication of microelectronics. Spiral wound membranes, another commonly employed technology, are readily affected by particles in the stream that restricts liquid flow paths through their extremely narrow channels. The fouling of polymeric ultra-filters is a major drawback as is the requirement to periodically clean these filters off line. [Sinha].